ABSTRACT The family of deubiquitinating enzymes (DUBs) perform key functions in virtually all physiological and patho- physiological processes. Importantly disruption of DUB function has been associated with a wide range of hu- man diseases, including cancer, infection, and neurodegeneration. However, there are currently few selective inhibitors of DUBs and no dedicated compound libraries focused on these important enzymes. The goal of our interdisciplinary team (Sara Buhrlage, DUB biology, chemical biology; Jarrod Marto protein bi- ochemistry, chemical proteomics) is to develop a robust screening platform, validated against our novel, fo- cused library of DUB-targeting compounds. In the fullness of time results from our proposal will drive develop- ment of new potent and selective covalent DUB inhibitors and study of DUB function in cellular and animal models. Consistent with PAR-17-438, we focus on assay development, with emphasis on incorporating multiple, or- thogonal biochemical assays as key validation steps. We will develop and validate our platform through three specific aims: Aim 1. To develop and execute a library-vs-library screen comprised of 49 DUBs and a focused collec- tion of 150 DUB-targeting compounds. We will use ubiquitin-based DUB activity probes (ABPs) in competi- tion format assays with compounds from our library of DUB-targeting compounds. Quantitative MS analysis of these assays will establish selectivity profiles of our compounds against cellular DUBs. We will use cell-based and intact protein CE-MS to confirm on-target activity and covalent binding. Aim 2. To map the proteome-wide reactivity of irreversible DUB-targeting compounds. We will use two novel mass spectrometry-based MS approaches to further confirm covalent target-ligand binding, as well as to identify the specific site of modification on each target. These assays will provide a direct readout of concentra- tion-dependent probe binding on DUBs as well as on potential off-target cysteines throughout the proteome. As a result, these assays will also serve as comprehensive counter screens for our compounds. Aim 3. To validate our platform's ability to streamline medicinal chemistry optimization campaigns. In this Aim we will validate the ability of our platform to quantify changes in selectivity for late-stage DUB probes refined through structure-activity relationship (SAR) studies. Data and results from our proposal will provide (i) a library of well-characterized covalent DUB compounds; (ii) a broadly-accessible resource with publicly available protocols and guidelines; (iii) a common bioanalytical framework applicable to other enzyme families; and (iv) hits for future development into selective probes and potential candidates for the NCI Experimental Therapeutics (NExT) Program.